A central challenge in biological research is to understand the organization and dynamics of the molecular machines underlying cellular function. Scanning transmission electron microscopy (STEM) of samples in liquid environments opens up the possibility of imaging molecular complexes in intact cels with nanometer resolution. Leveraging advances in micro-fluidics systems with electron-transparent windows and specific protein labels of nanoparticles, liquid STEM has the potential to offer much of the functionality of light microscopy with the high resolution of electron microscopy. The contrast mechanism in STEM permits the detection of nanoparticles with high atomic number (Z), such as gold, inside a several micrometer thick layer of low-Z material, such as water or cells. The use of fluorescent nanoparticles, such as quantum dots (QDs) that contain heavy atoms, allows liquid STEM images to be correlated efficiently with fluorescence images. The same technology platform can also be used for transmission electron microscopy (TEM) on thin cellular regions using modified microchips. In Phase I, we developed a prototype liquid STEM system, demonstrated the imaging of specifically labeled proteins on fixed whole cells in liquid with a spatial resolution of 4 nm, correlated fluorescence microscopy images with liquid STEM images, and recorded single-shot images of live yeast cells. In Phase II, we will develop and commercialize a liquid STEM system, refining both the STEM holder and the microchips for use with biological specimens. We will also optimize the system for use with light microscopes to further enhance its correlative microscopy capabilities. In addition to the effort to improve system hardware and microchips, we will also study the resolution of the liquid STEM technique within limits of radiation damage. The spatial resolution of liquid STEM depends on several factors. The electron dose is determined by the maximal allowed dose within the limit of radiation damage, and we will determine the STEM resolution achievable on the biological structure of fixed COS7 cells, on live COS7 cells, and on live yeast cells. Finally, with a team of leading collaborators, we have planned a series of experiments to further demonstrate that liquid STEM can be used to solve critical biological questions. We plan to utilize liquid STEM to examine the characteristic distribution of a representative lipid raft protein, cholera toxin B subunit (CTXB), across the cell surface. We will study cytokinesis in live yeast cells and, aiming to better understand the molecular mechanism of cytokinesis, we wil study the structural differences of a selection of mutants of live S. pombe yeast cells with liquid STEM. We will study the physical and chemical controls that organic matrices such as collagen and amelogenin play in directing the early stages of calcium phosphate nucleation in the context of both bone and tooth formation. Finally, we will, for the first time, characterize a wide variety of functionalized nanostructures intended for in vivo applications in their native environment and at atomic resolution. PUBLIC HEALTH RELEVANCE: Liquid STEM offers the potential to combine the ultra-high resolution of electron microscopy with much of the functionality of light microscopy. If successful, this proposal will develop and commercialize liquid STEM systems capable of imaging individual proteins in whole cells in water that can be used to study the functioning of cells. This novel microscopy technique will be significant for investigations of molecular processes in many fields of biomedical research, including cancer, virology, neuroscience, and cell biology.